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Phytochemistry 69 (2008) 1617–1624

PHYTOCHEMISTRY

Oligosaccharide polyester and triterpenoid saponinsfrom the roots of Polygala japonica

Jing Fu, Li Zuo, Jingzhi Yang, Ruoyun Chen, Dongming Zhang *

Institute of Materia Medica, Chinese Academy of Medical Sciences and Key Laboratory of Bioactive Substances and Resources Utilization of Chinese,

Herbal Medicine (Peking Union Medical College), Ministry of Education, 1 Xiannongtan Street, Beijing 100050, China

Received 30 October 2006; received in revised form 18 July 2007Available online 18 March 2008

Abstract

An oligosaccharide polyester, 1-O-(E)-p-coumaroyl-(3-O-benzoyl)-b-D-fructofuranosyl-(2 ? 1)-[6-O-(E)-feruloyl-b-D-glucopyranosyl-(1 ? 2)]-[6-O-acetyl-b-D-glucopyranosyl-(1 ? 3)-(4-O-acetyl)-b-D-glucopyranosyl-(1 ? 3)]-4-O-[4-O-a-L-rhamnopyranosyl-(E)-p-coumaroyl]-a-D-glucopyranoside (polygalajaponicose I), and four triterpenoid saponins, 3b, 23, 27-trihydroxy-29-O-b-D-glucopyranosyl-(1 ? 2)-b-D-glucopyranosyl-olean-12-en-28-oic acid (polygalasaponin XLVII), 3-O-b-D-glucopyranosyl presenegenin 28-O-a-L-rhamnopyranosyl-(1 ? 2)-b-D-fucopyranosyl ester (polygalasaponin XLVIII), 3-O-b-D-glucopyranosyl presenegenin 28-O-b-D-galactopyranosyl-(1 ? 5)-b-D-apiofuranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 4)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-glucopyranosyl ester (polygalasaponin XLIX)and 2b, 27-dihydroxy-3-O-b-D-glucopyranosyl 11-oxo-olean-12-en-23, 28-dioic acid 28-O-b-D-galactopyranosyl-(1 ? 5)-b-D-apiofurano-syl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 4)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-fucopyranosyl ester (polygalasaponin L), in addition to fiveknown compounds have been isolated from the roots of Polygala japonica.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Polygala japonica; Polygalaceae; Oligosaccharide polyester; Triterpenoid saponins; Polygalajaponicose; Polygalasaponin

1. Introduction

The genus Polygala (family Polygalaceae) consists ofmore than 500 species from all over the world, of which39 species are distributed throughout China. Some of thesespecies have been used as traditional Chinese medicines forthousands of years for treating amnesia, neurasthenia andinflammation. Polygala japonica Houtt., a perennial herba-ceous plant widely distributed in southern China, is such afolk herbal medicine and is used as an ataractic, an expecto-rant and an anti-inflammatory agent for pharyngitis. Theseactivities may be due to the presence of various saponins inP. japonica (Zhang et al., 1995a,b, 1996a,b), since pharma-

0031-9422/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.phytochem.2008.01.010

* Corresponding author. Tel.: +86 10 63165207; fax: +86 10 63165227.E-mail address: zhangdm@imm.ac.cn (D. Zhang).

ceutical studies have indicated that the saponins found inPolygala have antipsychotic (Chung et al., 2002) and expec-torant (Peng and Xu, 1998) effects. Oligosaccharide polyes-ters are another kind of typical constituents in the genusPolygala, and have been reported to have cognition improv-ing and cerebral protective effects (Kouin et al., 2004). Aspart of research into the bioactive constituents from thegenus Polygala, the chemical constituents from the rootsof P. japonica were investigated because previous workwas mainly concerned with the aerial parts (Zhang et al.,1995a,b, 1996a). This paper deals with the isolation andstructural elucidation of an oligosaccharide polyester andfour triterpenoid saponins, respectively, named polygalaja-ponicose I (1) and polygalasaponins XLVII-L (2–5), alongwith five known compounds, polygalasaponin XXVIII(6), polygalasaponin XXIV (7), polygalasaponin XXIX(8), sucrose (9) and 3-hydroxy-1,2,7-trimethoxyxanthone(10).

1618 J. Fu et al. / Phytochemistry 69 (2008) 1617–1624

2. Results and discussion

The Me2CO:EtOH (1:1) fraction of a 95% EtOH extractof P. japonica Houtt. was subjected to macroporous resin,silica gel column, MPLC and Sephadex LH-20 columnchromatographic purification steps, as well as semi-pre-parative scale HPLC to afford compounds 1–9. Compound10 was isolated from the EtOAc fraction of the 95% EtOHextract.

Polygalajaponicose I (1) was assigned a molecular for-mula of C75H90O40, deduced from the [M + H]+ ion m/zat 1631.5085 in the positive high resolution ESI MS(HR–ESI MS), as well as from analysis of the 13C NMRspectroscopic data (see Section 4). Its IR spectrum alsoindicated the presence of hydroxyl (3381 cm�1), carbonyl(1712 cm�1) and phenyl (1631, 1604, 1514, 835, 715 cm�1)groups. After acid hydrolysis of 1, the EtOAc portion con-tained benzoic acid, (E)-p-coumaric and (E)-ferulic acidmoieties, which were identified by comparing retentiontimes with those of authentic samples on HPLC; the aque-ous portion afforded D-glucose, D-fructose and L-rhamnose,identified by HPLC analysis of acyclic diastereoisomericderivatives of sugars.

The 1HNMR spectroscopic data showed five anomericprotons at d 6.45 (d, J = 2.5 Hz), 6.11 (brs), 5.20 (d,J = 8.0 Hz), 5.11 (2 H, d, J = 8.0 Hz), assigned to a-glu-cose, a-rhamnose, b-glucose, b-glucose and b-glucose,respectively; the 10 configurations were determined fromthe JH1�H2 values and by comparing the 13C NMR spectro-scopic data for C-3 and C-5 of rhamnose with the literaturedata for the a-orientation (Kasai et al., 1979). The 13CNMR spectrum of 1 indicated the presence of six anomeric(d 106.2, 105.6, 103.8, 103.4, 99.7, 92.6), two sets of acetyl,a set of benzoyl, a set of feruloyl, and two sets of p-couma-royl carbons. The sugar proton and carbon signals in theNMR spectra were assigned by a combined analysis ofNOE, 2D-NOESY, HSQC and HMBC experiments.

The linkage sites among the monosaccharides werededuced from the following correlations observed in theNOESY and NOE experiments: H-1 of Fru with H-1 ofGlc-1, H-1 of Glc-2 with H-2 of Glc-1, H-1 of Glc-3 withH-3 of Glc-1, H-1 of Glc-4 with H-3 of Glc-3, and H-1of Rha with H-3, H-5 of the set of p-coumaroyl protonsat d 7.33. These correlations indicated a pentasaccharideskeleton for 1a and a rhamnose linked to C-4 of a p-couma-royl group.

The HMBC spectrum further confirmed the above con-clusions because long-range correlations were also observedbetween the following protons and carbons in 1: H-4 of Glc-3 with a carbonyl carbon of acetyl (d 170.0), H-6 of Glc-4with a carbonyl carbon of acetyl (d 170.6), H-3 of Fru witha carbonyl carbon of benzoyl, H-6 of Glc-2 with a carbonylcarbon of feruloyl, H-4 of Glc-1 with a carbonyl carbon ofp-coumaroyl (d 166.33), and H-1 of Fru with a carbonylcarbon of p-coumaroyl (d 166.9). On the basis of these data,the structure of polygalajaponicose I (1) was established as1-O-(E)-p-coumaroyl-(3-O-benzoyl)-b-D-fructofuranosyl-

(2 ? 1)-[6-O-(E)-feruloyl-b-D-glucopyranosyl-(1 ? 2)]-[6-O-acetyl-b-D-glucopyranosyl-(1 ? 3)-(4-O-acetyl)-b-D-glu-copyranosyl-(1 ? 3)]-4-O-[4-O-a-L-rhamnopyranosyl-(E)-p-coumaroyl]-a-D-glucopyranoside (see Fig. 1).

Polygalasaponin L (2) had an [M + Na]+ ion m/z at1435.6013 in the positive HR–ESI MS, 14 mass unitshigher than that of the known compound 8

(1421[M + Na]+). Combined with analysis of the 1H and13C NMR spectra of 2, its molecular formula was deducedas C64H100O34. Comparison of the NMR spectroscopicdata of 2 and 8 showed that they were almost superimpos-able in terms of the sugar moieties but differed in the agly-cone moiety: 2 had a carbonyl carbon at dC 201.4 and theolefinic carbon signals were shifted downfield to dC 162.0,131.8 compared with the alkenyl resonances of 8 at dC

138.8, 127.8. The analysis of the HMBC spectrum indi-cated the carbonyl carbon (d 201.4) was at C-11 of the agly-cone (see Fig. 2). The structure of the sugar chain of 2 wasconfirmed by analysis of HMBC and NOE spectra. In theHMBC spectrum, long-range correlations were observedbetween the following protons and carbons: H-1 of Glcwith C-3 of aglycone, H-1 of Fuc with C-28 of aglycone,H-1 of Rha with C-2 of Fuc, and H-1 of Xyl with C-4 ofRha. Moreover, correlations were observed between H-1of Api and H-4 of Xyl and between H-1 of Gal and H-5of Api in the NOE experiment. Six monosaccharides inthe acid hydrolysate of 2 were identified as D-glucose,D-fucose, L-rhamnose, D-xylose, D-apiose and D-galactoseby the same methods as described for 1. Based on theabove evidence, the structure of 2 was elucidated as 2b,3b, 27-trihydroxy-3-O-b-D-glucopyranosyl 11-oxo-olean-12-en-23, 28-dioic acid 28-O-b-D-galactopyranosyl-(1 ?5)-b-D- apiofuranosyl-(1 ? 4)-b-D-xylopyranosyl-(1 ? 4)-a-L-hamnopyranosyl-(1 ? 2)-b-D-fucopyranosyl ester.

The following five saponins, polygalasaponin XLVIII(3), polygalasaponin XLIX (4), polygalasaponin XXVIII(6) (Zhang et al., 1996b), polygalasaponin XXIV (7)(Zhang et al., 1996a), and polygalasaponin XXIX (8)(Zhang et al., 1996b), contained the same aglycone knownas presenegenin, which was identified by comparison of theNMR spectroscopic data with the literature values (Toshioet al., 1995). Polygalasaponin XLVIII (3) gave a[M + Na]+ ion peak m/z at 995.4865 in the positive HR–ESI MS, 132 mass units less than that of 6 (1127[M + Na]+), implying the absence of a pentose moiety.The NMR spectroscopic data of compounds 3 and 6 werealmost identical except for a set of terminal xylose signals,indicating that 3 had a structure similar to 6 except that aproton of 3 was replaced by a xylose moiety in 6. TheNMR spectroscopic data for compounds 4 and 8 werealmost identical, except that 4 had one more set of glucosesignals than 8 and one less set of fucose resonances, indicat-ing that 4 had a structure similar to 8 except that the fucosein 8 was replaced by glucose in 4. In addition, the structureof 4 was confirmed by HMBC and NOE spectra. InHMBC, long-range correlations were observed betweenthe following protons and carbons: H-1 (dH 5.04) of Glc

CH2OH

COO

R2O

R1

R3

R4

R6

O

OH

OH

OH

HOO

O

OH

OH O

OHOH

O

O

OH

OH

O

O

OHOH

OO

OH

OH

HO

OH

-D-Glc -D-Fuc -L-Rha

-D-Xyl -D-Api -D-Gal

R1 R2 R3 R4 R5 R6

2

3

4

5

6

7

8

H

OH

OH

OH

OH

OH

OH

Glc

Glc

Glc

Glc

Glc

Glc

CH 2OH

COOH

COOH

COOH

COOH

COOH

COOH

H 2

H 2

H 2

H 2

H 2

H 2

=O

HH Glc

H

H

H

H

H

H

A

C

B

D

E

B

A =Rha(1 2) Fuc

D = Rha (1 2) FucXyl (1 4)

E = Rha (1 2) FucXyl (1 4) [Api(1 3)]

B = Api (1 4)Gal (1 5)

(1 5)

Rha (1 2)FucXyl (1 4)

C = Api(1 4)Ga Glcl Rha (1 2)Xyl (1 4)

OO

OHOH

OH

O

OHOH

OH

OH

O

OOH

OH

O

O

O

OH

OH

OH

OH

OOH

OH

OH

OH

1a

O

OHO

HO

O

OHOH

O

O

HO

OHO

OH

O

O

O

O

O

O

OHOH

OH

OHO

O

O

O

O

O

O

OH

OH

OH

OHO

O

O

Glc-2

1

2

Glc-1

OGlc-3

OHGlc-4

1

O

O

OHO

HO

O

OHOH

O

O

HO

OHO

OH

O

O

O

O

O

O

OHOH

OH

OHO

O

O

O

O

O

O

OH

OH

OH

OHO

O

O

Glc-2

1

2

Glc-1

OGlc-3

OHGlc-4

O

HMBC: H C NOESY: H H

β β β

(1 2)Glc-O-

Fig. 1. The structures of compounds 1–8 and key HMBC and NOE correlations of polygalajaponicose I (1).

J. Fu et al. / Phytochemistry 69 (2008) 1617–1624 1619

OO

OHOH

HO

C

O

O

O

O

OH

O

OHOH

OO

OH

OH

OO

OHOH

OO

OH

OH

HO

OH

COOH

CH 2OH

HO

O

OH

OH

11

9

12

30

HMBC: H C NOE: H H

Fig. 2. Key HMBC and NOE correlations of polygalasaponin L. (2).

1620 J. Fu et al. / Phytochemistry 69 (2008) 1617–1624

with C-3 of aglycone, H-1 (dH 6.23) of Glc with C-28 ofaglycone, H-1 of Rha with C-2 (dC 76.6) of Glc, and H-1of Xyl with C-4 of Rha. The NOE correlations wereobserved between H-1 of Api and H-4 of Xyl and betweenH-1 of Gal and H-5 of Api. Thus, polygalasaponin XLVIII(3) was determined as 3-O-b-D-glucopyranosyl presenege-nin 28-O-a-L -rhamnopyranosyl-(1 ? 2)-b-D-fucopyrano-syl ester, and polygalasaponin XLIX (4) was establishedas 3-O-b-D-glucopyranosyl presenegenin 28-O-b-D-galacto-pyranosyl-(1 ? 5)-b-D-apiofuranosyl-(1 ? 4)- b-D-xylopyrano-syl-(1 ? 4)-a-L-rhamnopyranosyl-(1 ? 2)-b-D-glucopyranosylester.

Polygalasaponin XLVII (5) displayed a [M + Na]+ ionpeak m/z at 851.4482 in the positive HR–ESI MS, corre-sponding to a molecular formula of C42H68O16. TheNMR spectroscopic data of 5 were characteristic of a tri-terpenoid glycoside with two sugar units. Detailed analysisof the NMR spectroscopic data established that theaglycone of 5 is 3b, 23, 27, 29-tetrahydroxy olean-12-en-28-oic acid (Hamburger and Hostettmann, 1986). Afteracid hydrolysis, 5 only afforded D-glucose sugar moieties,and the NMR spectroscopic data indicated the existenceof a b-D-glucopyranosyl-(1 ? 2)-b-D-glucopyranosyl group(Zhang et al., 1996b). The sugar chain of 5 was alsodeduced from the HMBC correlations between H-1 ofGlc-inn and C-29 of aglycone and between H-1 of Glc-ter and C-2 of Glc-inn. Therefore, the structure of polygal-asaponin XLVII was elucidated as 3b, 23, 27-trihydroxy-29-O-b-D-glucopyranosyl-(1 ? 2)-b-D-glucopyranosyl-olean-12-en-28-oic acid.

3. Conclusions

Compounds 1–5 are new derivatives belonging to theoligosaccharide and triterpenoid classes. Polygalajaponi-cose I (1) is one of the most structurely complex oligosac-charide polyesters found thus far. PolygalasaponinXLVII (5) shows an unusual substitution pattern of theD-12 oleanene skeleton, since in D-12 oleanene structures,the glucosyl moiety is usually linked to C-3 of the aglycone,and very few compounds have the sugar chain attached toC-29 of the aglycone (Hamburger and Hostettmann, 1986).Moreover, from our research and previous reports, it maybe concluded that the saponins found in the roots of Polyg-

ala japonica differ from those in the aerial parts: the sapo-nins found in the aerial parts of P. japonica have normalaglycones of D-12 oleanene type, while those found in rootsall have the hydroxyl group attached to C-27 of the agly-cones, which is an uncommon feature in olean-typetriterpenoids.

4. Experimental

4.1. General experimental procedures

Optical rotations were determined on a Perkin–Elmer241 digital polarimeter. Melting points were determinedon an XT-4 micro-melting point apparatus and are uncor-rected. IR spectra were recorded on an IMPACT 400 spec-trometer as KBr pellets. UV spectra were obtained on aShimadzu UV-260 spectrophotometer. 1H and 13C NMRspectra were acquired on a Varian Unity Inova spectrome-ter at 500 and 125 MHz, respectively, in pyridine-d5 atambient temperature using TMS as internal standard.The multiplicities of 13C NMR resonances were determinedby DEPT experiments. 1H–1H interactions were deter-mined by NOE and 2D NOESY. One-bond heteronuclear1H–13C connectivities were determined with 2D HSQC.Two- and three-bond heteronuclear 1H–13C connectivitieswere determined with 2D HMBC experiments, optimizedfor 2–3JCH of 8 Hz. ESI MS were measured on an Agilent1100 series LC/MSD Trap SL mass spectrometer andAutospee-Ultima ETOF. Reversed-phase HPLC was per-formed on a YMC-Pack ODS-A (YMC Co. Ltd.) column.Silica gel (100–200, 200–300 mesh, Qingdao) was used forCC and silica gel GF-254 (Qingdao) for TLC Analysis.Authentic samples were purchased from Sigma and Sinop-harm Chemical Reagent Co. Ltd. All chemical solventsused for isolation were of analytical grade or higher.

4.2. Plant material

The roots of Polygala japonica Houtt. were collected inJune 2003 in Jiangxi Province, China. The plant was iden-tified by Professor Yongming Luo, Jiangxi College of Tra-ditional Chinese Medicine. A voucher specimen wasdeposited in the Institute of Materia Medica, Chinese

J. Fu et al. / Phytochemistry 69 (2008) 1617–1624 1621

Academy of Medical Sciences and Peking Union MedicalCollege, Beijing (specimen number: 030621).

4.3. Extraction and isolation

The roots of P. japonica Houtt were air-dried after col-lection at ambient temperature, giving a dry weight of5.0 kg. The dried roots were pulverized and extracted with40 l of EtOH–H2O (95:5, v/v) by heating until reflux beganand maintaining this for 2 h. This process was repeated forthree times and a residue (0.839 kg) was obtained afterremoving the solvent under reduced pressure. This residuewas subjected to silica gel CC (100–200 mesh), eluted withCHCl3 (8 l), EtOAc (8 l), EtOAc:Me2CO (1:1, v/v, 8 l),EtOAc:Me2CO (1:3, v/v, 8 l), Me2CO (8 l), Me2CO:EtOH(1:1, v/v, 8 l), EtOH (8 l) and MeOH (8 l), successively.The Me2CO:EtOH (1:1, v/v) fraction (380 g) was appliedto D101 macroporous resin (2 kg) CC and eluted graduallyin this order: H2O (9 l), EtOH–H2O (3:7, v/v, 9 l), EtOH–H2O (6:4, v/v, 9 l) and EtOH–H2O (3:7, v/v, 9 l). TheEtOH–H2O (3:7, v/v) fraction (77.2 g) was subjected to asilica gel CC, and eluted with CHCl3:MeOH:H2O(70:30:5) to afford ten fractions and compound 9. Fractions5, 6 and 9 were separated by preparative scale MPLC witha mobile phase of MeOH–H2O mixture (4:6, 5;5, 6:4, 8:2,v/v), and every 1500 ml were sampled and their homogene-ity monitored by UV-detector with the absorbance wave-length at 210 nm, combining the fractions that showingsimilar UV-spectroscopic profiles. The samples were con-tinuously purified by Sephadex LH-20 using MeOH–H2Osolvents (1:9, 3:7, 6:4, 8:2, v/v, 500 ml) as gradient eluentand semi-preparative scale HPLC (MeOH:H2O, 50:50/53:47/55:45 + 0.05% TFA, v/v, 7 ml/min) to afford 1

(25 mg), 2 (30 mg), 3 (18 mg), 4 (22 mg), 5 (32 mg), 6

(27 mg), 7 (30 mg) and 8 (300 mg). The EtOAc extract(30.5 g) was subjected to silica gel CC (100–200 mesh), elut-ing with a gradient of CHCl3–MeOH (100:1 ? 100:25, v/v)to yield 61 fractions. Fractions 19–20 were separated bySephadex LH-20 CC (CHCl3–MeOH, 2:1, v/v) and recrys-tallized from MeOH to give 10 (4 mg).

4.4. Polygalajaponicose I (1)

White powder, ½a�14D �30.25 (MeOH; c0.40). ESI MS m/

z: 1631 [M + H]+, HR–ESI MS m/z: 1631.5085 [M + H]+

(calcd. for C75H91O40, 1631.5087). IR mKBrmax cm�1: 3381,

1712, 1631, 1604, 1514, 835, 715; UV kMeOHmax nm (log e):

208.0 (4.60), 228.0 (4.62), 306.6 (4.65). 1H NMR(500 MHz, C5D5N): d 8.40 (2H, d, J = 7.5 Hz; H-2, 6 ofbenzoyl), 8.02 (1H, d, J = 16.0 Hz; H-7 of feruloyl), 7.92(1H, d, J = 16.0 Hz; H-7 of p-coumaroyl-1), 7.79 (1H, d,J = 16.0 Hz; H-7 of p-coumaroyl-2), 7.67 (1H, m, H-4 ofbenzoyl), 7.65 (2H, d, J = 8.0 Hz; H-2, 6 of p-coumaroyl-2), 7.60 (2H, t, J = 7.5 Hz; H-3, 5 of benzoyl), 7.37 (1H,s; H-2 of feruloyl), 7.33 (2H, d, J = 8.0 Hz; H-3, 5 of p-cou-maroyl-1), 7.31 (2H, d, J = 8.0 Hz; H-2, 6 of p-coumaroyl-1), 7.30 (1H, d, J = 8.0 Hz; H-6 of feruloyl), 7.15 (1H, d,

J = 8.0 Hz; H-5 of feruloyl), 7.12 (2H, d, J = 8.0 Hz; H-3,5 of p-coumaroyl-2), 6.76 (1H, d, J = 16.0 Hz; H-8 of feru-loyl), 6.70 (1H, d, J = 16.0 Hz; H-8 of p-coumaroyl-1), 6.52(1H, d, J = 8.0 Hz; H-3 of Fru), 6.45 (1H, d, J = 2.5 Hz; H-1 of Glc-1), 6.38 (1H, d, J = 16.0 Hz; H-8 of p-coumaroyl-2), 6.11 (1H, brs; H-1 of Rha), 5.69 (1H, m; H-4 of Glc-4),5.46 (1H, t, J = 8.0 Hz; H-4 of Fru), 5.38 (1H, d,J = 9.0 Hz; H-5 of Glc-3), 5.34 (1H, d, J = 11.0 Hz; H-1aof Fru), 5.20 (1H, d, J = 8.0 Hz; H-1 of Glc-3), 5.14 (1H,d, J = 10.5 Hz; H-6a of Glc-2), 5.11 (2H, d, J = 8.0 Hz;H-1 of Glc-2; H-1 of Glc-4), 4.96 (1H, H-6b of Glc-2),4.94 (1H, H-1b of Fru), 4.74 (1H, H-5 of Fru), 4.67 (1H,H-3 of Glc-1), 3.82 (3H, s; H-OCH3 of feruloyl), 2.20(3H, s; H-AcO of Glc-3), 1.76 (3H, s; H-AcO of Glc-4),1.54 (3H, d, J = 6.0 Hz; H-6 of Rha). 13C NMR(125 MHz, C5D5N): d 170.6 [CO-AcO-(Glc-4)], 170.0[CO-AcO-(Glc-3)], 167.8 (C-9-feruloyl), 166.9 [C-9-(p-cou-maroyl-2)], 166.3 [C-9-(p-coumaroyl-1)], 166.3 (C-7-ben-zoyl), 161.4 [C-4-(p-coumaroyl-2)], 159.1 [C-4-(p-coumaroyl-1)], 151.0 (C-4-feruloyl), 148.9 (C-3-feruloyl),145.9 (C-7-feruloyl), 145.5 [C-7-(p-coumaroyl-2)], 144.8[C-7-(p-coumaroyl-1)], 133.7 (C-4-benzoyl), 130.7 [2C; C-2, 6-(p-coumaroyl-2)], 130.4 (2C; C-3, 5-benzoyl), 130.2[4C; C-2, 6-benzoyl, C-2, 6-(p-coumaroyl-1)], 129.1 (C-1-benzoyl), 128.7 [C-1-(p-coumaroyl-1), 126.7 (C-1-feruloyl),126.0 [C-1-(p-coumaroyl-2)], 123.9 (C-6-feruloyl), 117.4[2C, C-3, 5-(p-coumaroyl-1)], 116.7 [2C, C-3, 5-(p-couma-royl-2)], 116.6 (C-5-feruloyl), 116.5 [C-8-(p-coumaroyl-1)],115.2 (C-8-feruloyl), 114.7 [C-8-(p-coumaroyl-2)], 111.5(C-2-feruloyl), 106.2 [C-1-(Gcl-4)], 105.6 [C-1-(Glc-2)],103.8 [C-1-(Glc-3)], 103.4 (C-2-Fru), 99.7 (C-1-Rha), 92.6[C-1-(Glc-1)], 84.7 (C-5-Fru), 84.0 [C-3-(Glc-3)], 81.4 [C-2-(Glc-1)], 79.8 (C-3-Fru), 78.5 [C-3-(Glc-1)], 78.4 [C-3-(Glc-2)], 78.0 [2C; C-3, 5-(Glc-4)], 76.0 [C-2-(Glc-4)], 75.7[C-2-(Glc-2)], 75.0 [C-2-(Glc-3)], 73.6 (C-4-Rha), 73.1 (C-4-Fru), 72.5 [2C; C-3-Rha, C-5-(Glc-1)], 71.8 [C-5-(Glc-3)], 71.5 [C-4-(Glc-4)], 71.0 (C-5-Rha), 70.7 [C-5-(Glc-2)],70.5 [C-4-(Glc-1)], 69.3 [C-4-(Glc-3)], 63.5 [C-6-(Glc-2)],62.8 [C-6-(Glc-4)], 62.6 (C-6-Fru), 62.5 [C-6-(Glc-3)], 62.1[C-6-(Glc-1)], 55.9 (OCH3-feruloyl), 21.0 [CH3-AcO-(Glc-3)], 20.3 [CH3-AcO-(Glc-4)], 18.5 (C-6-Rha).

4.5. Polygalasaponin L (2)

White power, ½a�14D �5.56 (MeOH; c0.36). ESI MS m/z:

1435 [M + Na]+; HR–ESI MS m/z: 1435.6013 [M + Na]+

(calcd. for C64H100O34Na, 1435.5994). IR mKBrmax cm�1:

3406, 1678; UV kMeOHmax nm (log e): 204.0 (4.31). For 1H

and 13C NMR spectroscopic data, see Tables 1 and 2.

4.6. Polygalasaponin XLVIII (3)

White power, ½a�14D +11.30 (MeOH; c0.24). ESI MS m/z:

851 [M + Na]+; HR–ESI MS m/z: 851.4482 [M + Na]+

(calcd. for C42H68O16Na, 851.4405). IR mKBrmax cm�1: 3413,

1691; UV kMeOHmax nm (log e): 204.4 (4.01). For 1H and 13C

NMR spectroscopic data, see Tables 1 and 2.

Table 11H NMR spectroscopic data of compounds 2–5 (C5D5N, 500 MHz, dppm)*

2 3 4 5

Aglycone

2 4.69(1H, m) 4.71(1H, m) 4.70(1H, m) 1.762 – – – 1.823 4.55(1H, d,

3.0)4.59(1H, d, 3.0) 4.58(1H,

brs)4.08

12 6.32(1H, brs) 5.83(1H, t-like) 5.81(1H,brs)

5.87(1H, brs)

18 3.27(1H, dd,14, 3)

3.20(1H, dd, 14,4)

3.20(1H, d,12)

3.44(1H, d,14, 4)

24 1.96(3H, s) 1.96(3H, s) 1.93(3H, s) 1.02(3H, s)25 1.85(3H, s) 1.56(3H, s) 1.51(3H, s) 0.92(3H, s)26 1.34(3H, s) 1.12(3H, s) 1.11(3H, s) 1.02(3H, s)27 3.97(1H, d, 12) 3.71(1H, d, 12) 3.80(1H, d,

12)3.74

27 4.44(1H, d, 12) 3.91(1H, d, 12) 4.05(1H, d,12)

4.00(1H, d,12)

29 0.71(3H, s) 0.79(3H, s) 0.75(3H, s) 3.35(1H, d, 9)29 – – – 3.73(1H, d, 9)30 0.77(3H, s) 0.87(3H, s) 0.85(3H, s) 1.27(3H, s)

C-3 sugar

Glc-1 5.01(1H, d,7.5)

5.07(1H, d, 8.0) 5.04(1H, d,6.0)

2 3.92 3.90(1H, t, 6.0) 3.903 4.14 4.14 4.144 4.13 4.14 4.125 3.86 3.92 3.926 4.28 4.29 4.296 4.42 4.46 4.43

C-28 sugar

Fuc-1 6.03(1H, d,8.0)

6.04(1H, d, 8.0)

2 4.64(1H, t, 8.0) 4.74(1H, t, 8.5)3 4.13 4.164 3.93 3.955 3.91 3.906 1.45(3H, d,

6.0)1.48(3H, d,J = 6.0)

Glc-1 6.23(1H, d,7.5)

2 4.363 4.294 4.295 3.966 4.286 4.43

Rham-1

6.32(1H, brs) 6.53(1H, brs) 6.41(1H,brs)

2 4.80(1H, brs) 4.81(1H, brs) 4.80(1H,brs)

3 4.62 4.71 4.654 4.24 4.33 4.285 4.38 4.51 4.466 1.62(3H, d,

6.0)1.62(3H, d, 6.0) 1.71(3H, d,

6.0)

Xyl-1 4.86(1H, d,7.5)

4.89(1H, d,7.0)

2 4.04 4.023 4.02 4.024 4.13 4.14

Table 1 (continued)

2 3 4 5

5 3.34(1H, t, 11.0) 3.33(1H, t, 10.0)5 4.34 4.30

Api-1 6.36(1H, brs) 6.36(1H, brs)2 4.38 4.284 4.17 4.154 5.05(1H, d, 9.0) 5.055 4.18 4.155 4.66 4.67

Gal-1 4.83(1H, d, 7.5) 4.82(1H, d, 6.5)2 4.38 4.393 4.40 4.364 4.48 4.485 3.98 4.006 4.36 4.366 4.41 4.43

C-29 sugar

Glc-1 inn 4.80(1H, d, 7.5)2 4.153 3.954 4.155 4.216 4.066 4.35

Glc-1 ter 5.29(1H, d, 7.5)2 4.083 3.864 4.295 4.306 4.336 4.48

*Assignments were based on COSY, HSQC and HMBC experiments, anddue to severe overlapping in the 1H spectrum, only detectable relativeJ(Hz) are reported.

1622 J. Fu et al. / Phytochemistry 69 (2008) 1617–1624

4.7. Polygalasaponin XLIX (4)

White power, ½a�20D �12.16 (MeOH; c0.37). ESI MS m/z:

1437 [M + Na]+; HR–ESI MS m/z: 1437.6144 [M + Na]+

(calcd. for C64H102O34Na, 1437.6150). IR mKBrmax cm�1:

3413, 1678; UV kMeOHmax nm (log e): 204.2(4.26). For 1H

and 13C NMR spectroscopic data, see Tables 1 and 2.

4.8. Polygalasaponin XLVII (5)

Colorless power, ½a�20D +7.50 (MeOH; c0.16). ESI MS m/

z: 995 [M + Na]+; HR–ESI MS m/z: 995.48645 [M + Na]+

(calcd. for C48H76O20Na, 995.48276). IR mKBrmax cm�1: 3411,

1680; UV kMeOHmax nm (log e): 204.4 (4.46). For 1H and 13C

NMR spectroscopic data, see Tables 1 and 2.

4.9. Acid hydrolysis of compounds 1–5

To each compound (3 mg) was added 1 ml 10% HCl/H2O (v/v), with the resulting mixtures heated until refluxbegan at 75 �C. This was maintained for 3 h, with the mix-ture cooled and partitioned by EtOAc (1 ml � 3). EtOAcsolubles for each experiment was evaporated to dryness

Table 213C NMR spectroscopic data of compounds 2–8 (C5D5N, 125 MHz, d ppm)

2 3 4 5 2 3 4

Aglycone C-3 sugar

1 45.1 44.2 44.1 38.5 Glc-1 105.3 105.3 105.42 70.5 70.3 70.1 27.6 2 75.1 75.2 75.23 85.7 85.9 85.9 73.6 3 78.3 78.3 78.34 53.1 52.8 52.8 42.7 4 71.4 71.5 71.55 51.9 52.4 52.4 48.7 5 78.3 78.3 78.36 20.8 21.3 21.4 18.6 6 62.5 62.6 62.77 33.6 33.4 33.5 33.3 C-28 sugar

8 46.4 41.0 41.0 40.4 Fuc-1 94.9 94.99 63.2 49.3 49.3 48.7 2 74.3 73.910 37.5 37.0 36.9 37.3 3 76.5 76.911 201.4 23.4 24.4 24.1 4 73.1 73.312 131.8 127.8 127.8 127.8 5 72.5 72.413 162.0 138.9 138.9 139.5 6 16.8 16.914 49.5 48.0 48.0 47.8 Glc-1 94.715 25.1 24.5 23.9 24.3 2 76.616 23.1 24.4 23.5 23.8 3 79.317 46.6 46.8 46.9 46.8 4 71.218 42.4 41.9 41.9 40.8 5 78.819 43.3 45.3 45.2 40.3 6 61.920 30.5 30.7 30.7 35.6 Rham-1 101.4 101.4 101.321 33.5 33.8 33.8 29.1 2 71.7 72.3 71.722 31.6 33.2 33.3 32.4 3 72.2 72.5 72.423 180.7 180.6 180.7 68.3 4 85.1 73.4 84.824 14.0 14.2 14.1 13.0 5 68.3 69.7 68.325 18.8 17.5 17.4 16.3 6 18.4 18.6 18.526 21.2 18.7 18.5 18.8 Xyl-1 107.3 107.127 62.7 64.2 64.3 64.4 2 75.1 75.228 176.5 176.6 176.5 180.1 3 76.9 70.029 32.7 33.0 33.0 81.1 4 78.2 78.330 23.5 23.9 23.9 19.9 5 64.6 64.5C-29 sugar Api-1 109.4 109.4Glc-1 inn 103.4 2 77.8 77.72 83.7 3 81.3 81.33 78.5 4 76.1 76.14 71.3 5 67.6 67.65 77.9 Gal-1 103.1 103.26 62.8 2 72.5 72.2Glc-1 ter 106.3 3 73.0 73.22 76.9 4 70.1 70.33 78.2 5 77.0 76.84 71.5 6 62.0 62.15 77.96 62.6

J. Fu et al. / Phytochemistry 69 (2008) 1617–1624 1623

under reduced pressure at room temperature. The driedEtOAc fraction of 1 was next dissolved in MeOH andapplied to HPLC to compare retention times with thoseof authentic samples (YMC-Pack ODS-A, 150 � 4.6 mm,5 lm, CH3CN–H2O–TFA 450:1550:1, 254 nm, 35 �C, witha flow rate of 1.0 ml/min). As a result, (E)-p-coumaric acid(tR 4.25 min), (E)-ferulic acid (tR 4.85 min) and benzoicacid (tR 7.62 min) were detected. The dried H2O fractionof each compound was redissolved in H2O 0.2 ml, thenadded to a MeOH solution (0.2 ml) of L-(�)-a-methylben-zylamine (10 mg) and NaBH3CN (2 mg). The mixture wasallowed to stand overnight, acidified to pH 3 to 4 by addingglacial AcOH and evaporated to dryness. The resultant oilymaterial was acetylated by reaction with acetic anhydride-dry pyridine (1:1; 1 ml) at 100 �C for 1 h in a sealed tube.

After codistillation of the Ac2O with toluene, H2O (1 ml)was added to the residue and the mixture was extractedwith CHCl3. The CHCl3 layer was evaporated to drynessand purified by preparative TLC (CHCl3–MeOH 100:1),then applied to HPLC (ZORBAX SIL-100A, 5 lm,150 � 4.6 mm, n-hexane-EtOH 95:5, 230 nm, 30 �C, witha flow rate of 1.2 ml/min). The authentic samples were trea-ted by the same method and the retention times (tR) were:D-fructose 26.01 min, D-glucose 26.84 min, D-apiose 26.96min, L-rhamnose 29.30 min, D-fucose 32.98 min, D-xylose49.90 min, D-galactose 51.81 min. D-fructose was detectedfrom 1, D-glucose was detected from 1–5, D-apiose wasdetected from 2 and 4, L-rhamnose was detected from 1,2–4, D-fucose was detected from 2 and 3, D-xylose wasdetected from 2–4, D-galactose was detected from 2 and 4.

1624 J. Fu et al. / Phytochemistry 69 (2008) 1617–1624

Acknowledgements

Financial support for this research was provided by theNational Natural Science Foundation of China (No.20372087 204320307). We are grateful to the Departmentof Instrumental Analysis, Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking UnionMedical College for all spectra analyses.

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